DESCRIPTION: (Applicant's Abstract) It has been estimated that 40 percent of the brain synapses are GABAergic. Nevertheless, little is known about the the possible relationship between the GABAA receptor subunit composition and the synaptic and extrasynaptic localization of the receptor. The long-term objective of this proposal is to understand the molecular mechanisms involved in clustering and anchoring GABAA receptors at the postsynaptic membrane. The specific aims of this proposal are: 1) To test the hypothesis that the presence of gamma (gamma1, gamma2s, gamma2L or gamma3) subunits in the GABAAR pentamer in association with gephyrin and other synaptic proteins is necessary to cluster and position the GABAAR at GABAergic synapses; 2) To test the hypothesis that the alpha4 subunit in the hippocampus and thalamus associates with either the delta subunit or with a gamma subunit. The former GABAAR are extrasynaptic, the latter are synaptically localized; 3) To determine the cellular, subcellular, synaptic and extrasynaptic localization of beta1, beta2 and beta3, subunits in the rat brain in order to generate hypotheses about the possible relationships between beta subunit isoform and receptor localization and to test the hypothesis that the beta3 subunit is preferentially localized at synapses. Already available subunit-specific GABAA receptor antibodies in combination with immunocytochemistry at the light microscopy and EM levels will be used for revealing the cellular and subcellular localization of various GABAAR subunits at high resolution. We will do immunofluorescence in low density hippocampal cultures and immunogold at the EM level in intact rat hippocampus and thalamus and in mouse knockout mutants. Antisense treatment and transfections of primary hippocampal neuronal cultures will also be used to probe the mechanisms of subunit targeting to synapses. These studies will be relevant for understanding the mechanisms involved in the formation, maintenance and plasticity of the inhibitory GABAergic synapses in the brain. It is predicted that the inappropriate synaptic and extrasynaptic localization of the GABAA receptors, affects GABAergic synaptic function and brain development, leading to neurological and mental disorders.